RU2671974C2 - Method and system for determining biological response of target to soluble candidate substance - Google Patents

Abstract

FIELD: biology.SUBSTANCE: group of inventions relates to the detection of target-oriented drugs. Method for determining the biological response of a target to a soluble candidate substance comprises the following steps: introducing a soluble candidate substance into a laminar flow of a buffer liquid flowing through a dispersion channel to form a candidate substance solute in the buffer liquid having an initial concentration profile; by the laminar flow of the buffer liquid through the dispersion channel dispersing said candidate substance solute such that a dispersed concentration profile is formed in the buffer liquid; directing the laminar flow of the buffer liquid containing the candidate substance solute having said dispersed concentration profile into a detection channel to form a final symmetrical concentration profile of the candidate substance solute in the buffer liquid in the detection channel; introducing a target into the detection channel for mixing with the candidate substance solute, having a final symmetrical concentration profile in the buffer liquid to obtain in the buffer liquid a mixture having a combined concentration profile, the combined concentration profile comprising a target, having a constant target concentration profile, mixed with the candidate substance solute, having a final symmetrical concentration profile in the buffer liquid; hold the mixture of the target and the candidate substance solute in the buffer fluid in the detection channel so that at least one half of the combined concentration profile is retained in this channel; and performing optical scanning of the mixture of the target and the candidate substance solute contained in the buffer liquid retained in the detection channel and containing at least one half of the combined concentration profile, to detect, at various concentrations of the candidate substance solute contained in the combined concentration profile, an optical signal representing the biological response of the target to the soluble candidate substance. Also disclosed is a system for determining the biological response of a target to a soluble candidate substance.EFFECT: higher efficiency and reliability of determining the biological response of a target.15 cl, 3 tbl, 3 dwg

Description

The present invention relates to a method for determining the biological response of a target to a soluble candidate substance and the corresponding system according to the corresponding independent clause.

The detection of target-oriented drugs generally consists of rational drug design, chemical synthesis, biological analysis and data analysis, which are carried out repeatedly until a leader structure appears. Biological analyzes to determine the activity, selectivity, and effectiveness of a newly synthesized drug candidate against a target of interest are a fundamental part of this process.

A particular type of biological assay is the dilution method. In principle, the dilution method provides a dissolved candidate substance with different concentrations and is aimed at establishing a relationship between the biological response of the target and various concentrations of the dissolved candidate substance.

Application WO 2011/042509 discloses a method for creating a plurality of microdroplets with various concentrations of a solute (solute candidate) in a solvent (buffer liquid). Microdroplets also contain a target having a constant concentration. Microdrops are created by introducing a soluble candidate substance into the laminar flow of a solvent flowing through a microfluidic channel to form a dissolved candidate substance in a solvent. Immediately after introduction into the solvent, the dissolved substance has an initial concentration profile of a pulse shape. The laminar flow of the buffer fluid causes a change in the initial concentration profile of the dissolved substance in a pulse-like form in the solvent due to the Taylor – Aris dispersion into a dispersed concentration profile in the form of a Gaussian curve.

In this regard, it should be noted that the term "dispersed concentration profile having the shape of a Gaussian curve" as used in this application means a dispersed concentration profile, which theoretically has the shape of a Gaussian curve, however, in practice, the actual shape of the dispersed concentration profile may deviate slightly from the profile, having the shape of a regular Gaussian curve, in particular, this concerns the exact symmetry of the profile. Thus, whenever such a Gaussian-shaped profile is described as symmetrical and the actual dispersed concentration profile deviates slightly from a profile shaped as a regular Gaussian curve, the shape of the actual dispersed concentration profile may also deviate from the absolutely symmetrical shape.

Returning to the method described in WO 2011/042509, after introducing the target into a solvent containing a dispersed concentration profile in the form of a Gaussian curve, the continuous flow of the solvent is divided into many discrete microdrops. These microdrops are created by combining a continuous stream of solvent containing a dispersed concentration profile of a dissolved substance in the form of a Gaussian curve and a target having a constant concentration with the oil phase into a special stream, focusing the value so as to provide different average concentrations of dissolved substance in individual microdrops . The value of the step of the concentration of the dissolved substance is set by the difference in the average concentration in neighboring microdrops, which depends on the rate of formation of drops: if the rate of formation of drops decreases, the step size of the average concentration decreases.

Some disadvantages are associated with the described method for providing a target and a solute in a solvent in a series of microdrops. Microdrops share a concentration profile within discrete average concentrations (average concentrations in individual microdrops). This limits the number of different average concentrations (step size) to the number of microdroplets sharing the concentration profile, so that the "resolution" (step value) for various concentrations is limited. In addition, the use of oil to form microdrops precludes the use of candidate lyophilic substances or labels, because of their tendency to diffuse in oil.

Thus, it is an object of the present invention to provide a method for determining the biological response of a target to a soluble candidate substance that eliminates or at least substantially reduces the disadvantages known in the art.

The present invention provides a method for determining the biological response of a target to a soluble candidate substance, which comprises the following steps, in which:

- introducing a soluble candidate substance into the laminar flow of the buffer fluid flowing through the dispersion channel to form a dissolved candidate substance in the buffer fluid having an initial concentration profile;

- the laminar flow of the buffer liquid, the initial concentration profile of the dissolved candidate substance in the buffer liquid is dispersed through the dispersion channel to form a dispersed concentration profile of the dissolved candidate substance in the buffer liquid;

- direct the laminar flow of the buffer fluid containing the dissolved candidate substance having a dispersed concentration profile into the detection channel to form the final symmetric concentration profile of the dissolved candidate substance in the buffer liquid in the detection channel;

- introduce the target into the detection channel so as to obtain a combined concentration profile in the buffer fluid containing a constant concentration profile of the target lying on top of the final symmetric concentration profile of the dissolved candidate substance;

- at least one half of the combined concentration profile contained in the buffer fluid is held in the detection channel; and

- make an optical scan of at least one half of the combined concentration profile contained in the buffer fluid held in the detection channel to detect, at various concentrations of the dissolved candidate substance, the combined concentration profile, an optical signal representing the biological response of the target to the soluble candidate substance.

Accordingly, the method according to the invention assumes that at least one half of the combined concentration profile is held in the detection channel in the form of a stationary continuous profile, so that, in principle, an unlimited number of detection points can be selected for optical scanning along the entire profile (restrictions are only associated with optical scanner resolution). A stationary continuous profile allows optical scanning of the combined concentration profile for a given period of time, during which the combined concentration profile is stable due to the fact that the (molecular) diffusion processes during this given period of time are negligible. The combined concentration profile of the dissolved candidate substance is contained only in the buffer liquid (not in the oil phase) so that candidate lipophilic substances or targets can also be used in the analysis.

The described method is in principle applicable to known dilution methods in which the activity, selectivity and effectiveness of a soluble candidate substance are typically determined with respect to a target of interest. “Biological response of a target to a soluble candidate substance” as used in this application includes any biological, biochemical, pharmaceutical, etc. a response suitable for creating a dosage-response curve using the dilution method. The following tables, including analysis examples, are classified according to various indications that correspond to the biological response of the target to the soluble candidate substance.

Fluorescence

Luminescence

Other indications

As an example, a target may refer to a group consisting of proteins (soluble proteins, membrane proteins), such as enzymes (e.g., kinases, proteases, peptidases), transport proteins (e.g., ion channels, albumin), receptors conjugated to G β-protein (e.g., histamine receptor, serotonin receptor), transcription factors, etc.

By soluble candidate substance is meant a substance to which a target of interest can respond. For example, a soluble candidate substance may be an active ingredient that is soluble in a buffer fluid per se, or a soluble candidate substance may be an active ingredient (e.g., a pharmaceutically active substance) that is already dissolved in a solvent (e.g., a solution containing an active ingredient) and a solution containing the active ingredient is soluble in the buffer liquid. The term “soluble” refers to the ability of a particular soluble candidate substance to dissolve in the appropriate buffer fluid used for a particular assay. The term “buffer fluid” refers to any suitable known fluid that is generally inert with respect to a determined biological response. The term “target” as used in this application includes individual substances as well as “combined targets” as described below.

The buffer fluid can be held in the detection channel by any suitable measure, as a result of which the laminar flow of the buffer liquid is stopped so as to stop the dispersion effects associated with the Taylor – Aris dispersion in the detection channel. During the period of time required for optical scanning, other molecular (diffusion) processes can be neglected, and the combined concentration profile contained in the buffer liquid and permanently held in the detection channel can be considered stable.

The dispersion channel and the detection channel may be a microfluidic channel having an inner diameter of less than 2 mm, more preferably less than 1 mm, and most preferably less than 100 microns. It is necessary that both the dispersion channel and the detection channel allow the passage of a laminar flow of buffer fluid, which requires an appropriate Reynolds number. The laminar flow of the buffer liquid determines the initial concentration profile of the dissolved candidate substance, which changes due to the Taylor – Aris dispersion (see description above). The Taylor-Aris dispersion causes a change in the concentration profile of the dissolved candidate substance to a concentration profile in the form of a Gaussian curve.

In one embodiment of the method of the invention, the step of holding at least one half of the combined concentration profile contained in the buffer fluid in the detection channel is performed by stopping the subsequent introduction of the buffer liquid into the dispersion channel and the target into the detection channel. In a practical example, the subsequent supply of the buffer fluid is stopped by shutting off the feed pump, so as to stop the flow of the laminar fluid. Such a pump can be time-controlled so that it can automatically shut off after a given period of time. This period of time begins from the moment the laminar flow of the buffer fluid containing the dispersed concentration profile of the dissolved candidate substance enters the detection channel and ends when at least one half of the combined concentration profile is completely inside the detection channel. Similar considerations apply to stopping the supply of the target to the detection channel, which can be achieved by turning off the target supply pump, which introduces the laminar flow of the target into the detection channel.

According to an additional variant of the method according to the invention, a laminar flow of a buffer liquid containing a dissolved candidate substance having a dispersed concentration profile is directed to the detection channel at a constant flow rate. Also, the target is introduced into the detection channel at a constant flow rate to obtain a combined concentration profile in the buffer fluid containing a constant concentration profile of the target lying on top of the final symmetric concentration profile of the dissolved candidate substance. This is a practical approach that allows one to achieve in the detection channel a very uniform coating of the final symmetric concentration profile of the dissolved candidate substance in the buffer liquid with a constant target concentration profile. From a practical point of view, the introduction of the target at a constant flow rate begins even before the final concentration profile of the candidate solute in the buffer liquid reaches the detection channel, so that the laminar target flow is introduced at the point in time when the final concentration profile of the candidate candidate solute in the buffer fluid reaches the detection channel. This laminar target flow then smoothly lays on top of the final concentration profile of the candidate substance to form a combined concentration profile in the detection channel.

According to a further embodiment of the method according to the invention, the step of holding at least one half of the combined concentration profile contained in the buffer liquid in the detection channel comprises holding only one half of the combined concentration profile in the detection channel. Although, in general, the entire combined concentration profile or more than one half of the combined concentration profile can be held in the detection channel and scanned, it is necessary to keep only one half of the combined concentration profile in the detection channel, since one half of the combined concentration profile contains all the concentrations of interest . Thus, the length of the detection channel can be reduced, and the scanning time of the combined concentration profile in the detection channel (in a given resolution) can also be reduced, since it is necessary to optically scan only one half of the combined concentration profile, and not the entire combined concentration profile. This can be of great importance, in particular, due to the large number of analyzes performed at an early stage of the search for drugs, which should be largely automated if possible.

According to another additional variant of the method according to the invention, only one half of the combined concentration profile contains at least five (preferably from five to six) orders of magnitude of the concentration of the dissolved candidate substance in the buffer fluid. Five orders of magnitude of the concentration of the dissolved candidate substance in the buffer liquid includes a range from 1 to 100'000 nM [nano-Molar], six orders of magnitude a range from 1 to 1'000'000 nM. This proves that only one half of the combined symmetrical concentration profile is sufficient to detect (if present) any significant biological response.

According to a further embodiment of the method according to the invention, the step of optical scanning in the detection channel of at least one half of the combined concentration profile contained in the buffer liquid is performed by moving the detection channel relative to a stationary optical detection unit. As a rule, only the relative movement of at least one half of the combined concentration profile contained in the buffer fluid in the detection channel is sufficient to scan at least one half of the combined concentration profile to obtain a signal characteristic of the biological response of the target to the soluble substance -candidate. However, since high resolution optical systems are typically very sensitive to any changes, it is preferable to position the optical detection unit stationary while moving the detection channel (for example, a chip containing the detection channel) relative to the optical detection unit.

According to an additional variant of the method according to the invention, the step of optical scanning in the detection channel of at least one half of the combined concentration profile contained in the buffer liquid is performed by repeatedly moving the detection channel in the same range of relative locations of the detection channel and an optical detection unit, where corresponding signals representing various biological responses are then converted to form an average signal and and signal change according to time, which are characteristic for the target biological response to soluble candidate substance. Obtaining a number of separate signals for each concentration of the combined concentration profile and averaging these signals allows us to obtain a signal that further reflects the biological response.

According to another additional variant of the method according to the invention, the stage of optical scanning in the detection channel of at least one half of the combined concentration profile contained in the buffer fluid is performed at different detection sensitivity by adjusting the sensitivity of the optical detection unit to the optical signal, which represents the biological response of the target to a soluble candidate and / or to an optical signal representing the concentration of soluble thing candidate society.

In principle, any optical reader may be used as an optical detection unit for reading a corresponding characteristic optical signal. For example, the optical detection unit may be a CCD camera, which is typically sensitive to light intensity corresponding to 2-3 orders of magnitude of the concentration of the dissolved candidate substance, although the range of scanned concentrations typically covers 5-6 orders of magnitude of the concentration of the dissolved candidate substance. Thus, it is preferable to change the detection sensitivity so that it is possible to use the same CCD camera to scan the combined concentration profile over the entire range where a signal characteristic of the biological response of the target can be expected.

According to a still further embodiment of the method according to the invention, the target introduced into the detection channel is a combined target containing at least two components that are separately introduced into the detection channel. For example, if the candidate substance is a substance that is analyzed to determine its activity to inhibit the converting activity of the enzyme, then the combined target should contain not only the enzyme, but also a component transformed by the enzyme in the absence of an inhibitor.

Accordingly, the combined target contains two components. In this case, the biological response will be the activity of one component (converting enzyme) of the combined target to transform another component of the combined target. Both components of the combined target can be separately introduced into the detection channel so as to create a combined concentration profile in the detection channel. This embodiment can be used, for example, to determine the concentration of half-maximal inhibition (IC ₅₀ ) to determine the activity of a candidate substance to inhibit the converting activity of the enzyme.

According to a further embodiment of the method of the invention, either a soluble candidate substance that forms a dissolved candidate substance, or a target or both contain a fluorescent marker that is capable of emitting an optically detectable fluorescent signal. If the fluorescent marker is attached to the candidate substance (for example, if the concentration gradient cannot be easily detected otherwise), the optically detectable fluorescent signal emitted by the fluorescent marker allows the actual concentration of the soluble candidate substance to be detected in the detection channel within at least of at least one half of the concentration profile contained in the buffer liquid, thus, it becomes possible to determine the localization of individual concentrations in the detection channel s within at least one half of the concentration profile contained in the buffer liquid. Calibration (ex situ in another experiment, or in situ in the same experiment) can be performed in this way. If the target contains a fluorescent marker, after calibrating the intensity of the signal emitted by the fluorescent marker, it can be used as an optical signal representing the biological response of the target to a soluble candidate. For example, if a component that can be converted by an enzyme contains a fluorescent marker, converting the component can reduce luminescence from the substrate. In the event that both the dissolved candidate substance and the target contain a fluorescent marker, it is obvious that the luminescent radiation of the marker of the dissolved candidate substance and the luminescent radiation of the target marker must have different wavelengths in order to distinguish between the luminescent radiation emitted by the marker of the dissolved candidate substances and luminescent radiation of the target marker.

Another aspect of the present invention relates to a system for determining the biological response of a target to a soluble candidate substance, comprising:

a dispersion channel having

the first entrance to the dispersion channel for introducing a buffer liquid into the dispersion channel,

a second entrance to the dispersion channel, located further than the first entrance to the dispersion channel, for introducing a soluble candidate substance into the buffer fluid flowing through the dispersion channel to form a dissolved candidate substance in the buffer liquid, and

the exit from the dispersion channel, located further than the first and second entrances to the dispersion channel, allowing the buffer fluid containing the dissolved candidate substance to exit the dispersion channel,

- a pump for creating a laminar flow of a buffer fluid through a dispersion channel,

an injector of a candidate substance for introducing a soluble candidate substance into a laminar flow of a buffer fluid through a dispersion channel, to form a dissolved candidate substance in a buffer fluid having an initial concentration profile, which is then dispersed by a laminar flow of a buffer fluid through a dispersion channel to form a dispersed concentration profile a candidate solute in a buffer liquid,

- a detection channel having:

the first entrance to the detection channel, which is arranged to communicate in fluid with the exit from the dispersion channel so that the laminar flow of the buffer liquid exiting the dispersion channel through the exit from the dispersion channel and containing the dispersed concentration profile of the dissolved candidate substance is directed through the first entrance of the channel detection in the detection channel for the formation of the final symmetric concentration profile of the dissolved candidate substance in the buffer fluid in the channel Detection and

at least one additional input to the detection channel for introducing the target into the detection channel,

- at least one target injector for introducing the target into the detection channel through at least one additional entrance to the detection channel so as to obtain a combined concentration profile containing a constant concentration profile of the target lying on top of the final symmetric concentration profile of the dissolved substance buffer fluid candidate

- means for holding in the detection channel at least one half of the combined concentration profile contained in the buffer fluid, and

- an optical detection unit capable and located for optical scanning of at least one half of the combined concentration profile contained in the buffer fluid in the detection channel for detecting, at various concentrations of the dissolved candidate substance, the combined concentration profile, an optical signal representing the biological response of the target to the soluble candidate substance

in which the output from the dispersion channel and the first entrance to the detection channel are connected to each other so that the laminar flow is maintained at the connection of these channels and in the detection channel.

According to one embodiment of the system of the invention, the inner wall of the dispersion channel at the outlet of the dispersion channel and the inner wall of the detection channel at the entrance to the detection channel have the same shape and size to provide a continuous contour of the inner wall of the channel at the connection of the dispersion channel and the detection channel.

According to another embodiment of the system of the invention, the inner wall of the dispersion channel at the outlet of the dispersion channel and the inner wall of the detection channel at the entrance to the detection channel are integrally formed so as to form a common continuous inner wall.

Further preferred embodiments of the invention will become apparent from the following description of the invention with reference to the accompanying drawings, in which:

FIG. 1 shows a cross-sectional view of a dispersion channel and a detection channel, which are made integrally in the process of the invention; and

FIG. 2 shows a perspective view of an embodiment of the system of the invention in which the detection channel is located on a chip movable relative to the optical detection unit, and

FIG. 3 shows an enlarged image of the element III shown in FIG. 2

FIG. 1 shows a dispersion channel 11 and a detection channel 12, which are integrally formed. The dispersion channel 11 has a first input 111 into the dispersion channel, and a second input 112 into the dispersion channel, and an exit 113 from the dispersion channel. The second input 112 to the dispersion channel is located further than the first input 111 to the dispersion channel, and the exit 113 from the dispersion channel is located further than the first and second inputs 111, 112 to the dispersion channel. The detection channel 12 has an input 121 to the detection channel, which is identical to the output 113 from the dispersion channel, and two additional inputs 122, 123.

In action, the buffer liquid 2 is introduced into the dispersion channel 11 through the first inlet 111 into the dispersion channel through a pump 1 (see FIG. 2), and the parabolic velocity profile of the laminar flow of the buffer liquid 2 flowing through the dispersion channel 11 is shown by arrows. The injector 7 of the candidate substance is located at the second inlet 112 into the dispersion channel for introducing the soluble candidate substance into the laminar flow of the buffer fluid 2 flowing through the dispersion channel 11. In the shown embodiment, the soluble candidate substance is a soluble candidate substance capable of inhibiting conversion enzyme activity. Immediately after the introduction of the buffer liquid into the laminar flow, the soluble candidate substance is dissolved in the buffer liquid 2 to form the dissolved candidate substance 3 in the buffer liquid 2 having an initial concentration profile, schematically shown as a rectangular profile 31 shown above the second entrance to the dispersion channel although the actual concentration profile is not rectangular as shown.

As already described above, the initial concentration profile 31 is then dispersed due to the Taylor-Aris dispersion, under the action of the laminar flow of the buffer liquid 2 in the dispersion channel 11 so that the initial concentration profile 31 changes to the dispersed concentration profile shown in the form of the Gaussian curve 32 shown above exit 113 from the dispersion channel 11. A buffer liquid 2 containing a dispersed concentration profile 32 is then sent to the detection channel 12 through the entrance 121 to the channel 12 obn the environment, which in the shown embodiment is identical to the exit 113 from the dispersion channel 11, since the dispersion channel 11 and the detection channel 12 are integrally formed so that the inner wall 13 of the dispersion channel 11 at the exit 113 of the dispersion channel and the inner wall 14 of the detection channel 12 the first entrance 121 to the detection channel have the same shape and size to provide a continuous inner wall of the channel at the connection of the dispersion channel 11 and the detection channel 12. This allows the buffer fluid 2 to be moved from the dispersion channel 11 to the detection channel 12, while maintaining the laminar flow so that the dispersed concentration profile 32 is then dispersed in the detection channel 12 to form the final symmetric concentration profile represented by the Gaussian curve 33 shown above the detection channel 12.

The detection channel 12 contains two additional inputs 122, 123 to the detection channel, which are located at the connection of the dispersion channel 11 to the detection channel 12. Two components 41, 42 of the target are separately introduced into the detection channel through two additional inputs 122, 123 using two injectors 8, 9 of the target (see Fig. 2). In practice, the flow of the drilling fluid 2, as well as the flows of the combined target containing two components 41, 42 of the target (for example, two separate solutions of the liquid target) are continuously fed until the soluble candidate substance is introduced into the dispersion channel 11. This ensures a constant concentration of the combined target containing two components 41, 42 in the detection channel 12. This constant profile of the combined target lies on top of the final symmetric concentration profile 33 of the dissolved candidate 3 in the detection channel 12 to form a combined concentration profile containing a constant concentration profile of the target lying on top of the final symmetric concentration profile 33 of the dissolved candidate.

As already discussed above, the introduction of a combined target containing two components 41, 42 allows you to perform special biological analyzes with combined targets. Thus, the combined target may contain, for example, enzyme 41 and component 42, which can be converted by the action of the enzyme. Enzymatic activity as a biological response can be determined by detecting the conversion rate of component 42. If the candidate substance is an inhibitor of the enzyme that inhibits the activity of the enzyme 41 to conversion, a decrease in enzymatic activity should be a biological response.

As soon as at least one half of the combined concentration profile containing a constant target concentration profile lying on top of the final symmetric concentration profile 33 of the dissolved candidate 3 substance enters the detection channel 12, it is held there. As already explained above, one half of the combined concentration profile is sufficient due to the symmetry of the combined concentration profile. Of course, it is possible to retain the entire combined concentration profile in the detection channel 12. A buffer liquid 2 containing at least one half of the combined concentration profile is held in the detection channel by stopping the subsequent supply of the buffer liquid 12, as well as the supply of the target components 41, 42 to the detection channel 12. The termination of the laminar flow leads to the termination of the Taylor - Aris dispersion, while other (molecular) diffusion processes can be neglected for the period of time necessary to complete the stage of optical scanning of at least one half of the combined concentration profile held in the detection channel. In the present example, the biological response can be detected by optical detection of the fluorescent signal emitted by the fluorescent marker contained in component 42. The conversion of component 42 under the action of the enzyme leads to a decrease in the intensity of the detected fluorescent signal (a decrease can be detected in analysis methods based on quenching effects). This decrease in the detectable fluorescent signal is a consequence of enzymatic activity. Accordingly, if the candidate substance is an inhibitor of the enzyme that inhibits the activity of the enzyme towards the conversion of component 42, the detected fluorescence signal from component 42 will either not decrease at all, or it will decrease slightly. Accordingly, in this example, an optically detectable signal representing a biological response is a change in the intensity of the luminescent radiation emitted by the fluorescent marker contained in component 42.

This reduced intensity reduction can be determined within at least one half of the combined concentration profile held in the detection channel 12. Since at least one half of the combined concentration profile contains continuous “dilutions” in at least one half of the combined concentration profile (different concentrations of the candidate 3 solute in the buffer fluid, ideally covering the entire concentration range from zero to the initial concentration, however, according to at least five to six orders of magnitude), the dilution method allows you to determine the biological response in the entire range of almost all concentrations of dissolved in exists candidate 3, contained in the combined concentration profile.

Luminescent radiation can be detected along the channel using a CCD camera 6 (see FIG. 2) used as an optical detection unit. The CCD camera detects luminescent radiation intensity at various locations of the detection channel 12 relative to the CCD camera.

An embodiment of the system of the invention is shown in FIG. 2. The system can be used, for example, to implement the method described above in connection with FIG. 1. In this embodiment of the system of the invention, portions of the dispersion channel 11 and the detection channel 12 are located on the chip 5. The chip 5 is moveable, which allows you to change the location of the various parts of the detection channel 12 relative to the CCD of the camera 6 used as an optical detection unit. Only the portion of the dispersion channel 11 is located on chip 5, while the dispersion channel 11 also contains a capillary 114 designed to increase the length of the dispersion channel to provide sufficient dispersion of the initial concentration profile 31 due to the Taylor-Aris dispersion. The pump 1 is located at the inlet 111 to the dispersion channel for injecting the buffer fluid into the dispersion channel 11 and for causing a laminar flow through it. The second inlet 112 to the dispersion channel is located farther from the first inlet 111 to the dispersion channel, where the soluble candidate substance is introduced into the buffer liquid using the injector 7 of the candidate substance. Using pump 1, a laminar flow is generated through a capillary 114 and through a portion of the dispersion channel 11 located on the chip as described above, the dispersion of the initial profile of the candidate solute is represented by a rectangular curve 31, the formation of a dispersed profile due to Taylor-Aris dispersion is shown in the form of a Gaussian curve 32 (see Fig. 1). The output 113 from the dispersion channel, as well as two additional inputs 122, 123 to the detection channel, are located on the chip 5. Two additional inputs 122, 123 to the detection channel are combined with the first input 121 to the detection channel, which is identical to the output 113 from the dispersion channel (see Fig. 3). The detection channel 12 contains a section of bending shape located on the chip 5. The output 125 from the detection channel is located at the end of the detection channel 12 for leakage of buffer fluid from the detection channel 12.

Embodiments of the invention have been described using the drawings. However, various modifications and variations of the described embodiments are possible without departing from the general idea underlying the present invention. Therefore, it should not be understood that the invention is limited by the described embodiments of the invention, but rather by the scope of the claims defined by the claims.

Claims (34)

1. A method for determining the biological response of a target (41; 41, 42) to a soluble test substance, comprising the following steps: - a soluble test substance is introduced into the laminar flow of the buffer liquid (2) flowing through the dispersion channel (11) with the formation of the dissolved test substance (3) in the buffer liquid (2) having an initial concentration profile (31); - by a laminar flow of the buffer liquid (2) through the dispersion channel (11), the dissolved test substance (3) having an initial concentration profile (31) in the buffer liquid (2) is dispersed so that the dispersed concentration profile is contained in the buffer liquid (2) ( 32); - direct the laminar flow of the buffer liquid (2) containing the test substance solution (3) having a dispersed concentration profile (32) into the detection channel (12) so that in the detection channel (12) the dissolved test substance (3) forms a final symmetric the concentration profile (33) of the dissolved test substance (3) in the buffer liquid (2); - introduce the target (41; 41, 42) into the detection channel (12) for mixing with the dissolved test substance (3) having a finite symmetric concentration profile (33) in the buffer liquid (2) so as to obtain in the buffer liquid (2 ) a mixture having a combined concentration profile, wherein the combined concentration profile contains a target (41; 41, 42) having a constant target concentration profile mixed with a dissolved test substance (3) having a finite symmetric concentration profile (33) in a buffer liquid ( 2); - hold the mixture of the target (41; 41, 42) and the dissolved test substance (3) in the buffer liquid (2) in the detection channel (12) so that at least one half of the combined concentration profile is retained in the detection channel (12); and - make an optical scan of the mixture of the target (41; 41, 42) and the dissolved test substance (3) contained in the buffer liquid (2) held in the detection channel (12) and containing at least one half of the combined concentration profile to detect at various concentrations of the solution (3) of the test substance contained in the combined concentration profile, an optical signal representing the biological response of the target (41; 41, 42) to the soluble test substance. 2. The method according to claim 1, wherein the step of holding in the detection channel (12) a mixture of the target (41; 41, 42) and the dissolved test substance (3) in the buffer liquid (2) held in the channel (12) detection and containing at least one half of the combined concentration profile is performed by stopping the subsequent introduction of the buffer fluid (2) into the dispersion channel (11) and the target into the detection channel. 3. The method according to claim 1, in which the laminar flow of a buffer liquid (2) containing a dissolved test substance (3) having a dispersed concentration profile (32) is sent to the detection channel (12) at a constant flow rate, and in which the target (41; 41, 42) is introduced into the detection channel (12) at a constant flow rate to obtain a mixture having a combined concentration profile containing a target (41; 41, 42) having a constant target concentration profile (41; 41, 42), mixed with a dissolved test substance (3) having a finite th symmetric concentration profile (33) of the solution (3) of the test substance in the buffer liquid (2). 4. The method according to claim 2, in which the laminar flow of a buffer liquid (2) containing a dissolved test substance (3) having a dispersed concentration profile (32) is sent to the detection channel (12) at a constant flow rate, and in which the target (41; 41, 42) is introduced into the detection channel (12) at a constant flow rate to obtain a mixture having a combined concentration profile containing a target (41; 41, 42) having a constant target concentration profile (41; 41, 42), mixed with a dissolved test substance (3) having a finite th symmetric concentration profile of the solution (3) of the test substance. 5. The method according to any one of paragraphs. 1-4, where the stage at which a mixture of the target (41; 41, 42) and the dissolved test substance (3) in the buffer liquid (2) is held in the detection channel (12) so that at least one half of the combined concentration profile held in the detection channel (12), contains holding only one half of the combined concentration profile (33) in the detection channel (12). 6. The method according to p. 5, in which the mixture of the target (41; 41, 42) and the dissolved test substance (3) in a buffer liquid (2) held in the detection channel (12) and containing only one half of the combined concentration profile (33 ), contains at least five orders of magnitude of the concentration of the dissolved analyte (3) in the buffer liquid (2). 7. The method according to any one of paragraphs. 1-4, in which the stage at which optical scanning is performed in the channel (12) for detecting a mixture of the target (41; 41, 42) and the dissolved test substance (3) contained in the buffer liquid (2) held in the channel (12) detection and containing at least one half of the combined concentration profile is performed by moving the detection channel (12) relative to a stationary optical detection unit (6). 8. The method according to claim 7, in which the stage of optical scanning in the channel (12) for detecting a mixture of the target (41; 41, 42) and the dissolved test substance (3) contained in the buffer liquid (2) held in the detection channel (12) and containing at least one half of the combined concentration profile, is performed by repeatedly moving the detection channel (12) in the same range of relative positions of the detection channel (12) and the optical detection unit (6), and in which the corresponding signals represent s various biological responses, then converted to form an average signal level, or signal a change in function of time, which are target biological response to the soluble analyte. 9. The method according to p. 7, in which the stage at which the optical channel scans a mixture of the target (41; 41, 42) and the dissolved test substance (3) contained in the buffer fluid (2) held in the channel (12) ) detection and containing at least one half of the combined concentration profile, is performed at different detection sensitivity by adjusting the detection sensitivity of the optical detection unit (6) to an optical signal representing the biological response of the target (41; 41, 42) to the solution My test substance, and / or optical signal representing the concentration of the soluble analyte. 10. The method according to p. 8, in which the stage at which an optical scan of the mixture of the target (41; 41, 42) and the dissolved test substance (3) contained in the buffer liquid (2) held in the channel (12) is carried out in the detection channel ) detection and containing at least one half of the combined concentration profile, is performed at different detection sensitivity by adjusting the detection sensitivity of the optical detection unit (6) to an optical signal representing the biological response of the target (41; 41, 42) to the solution a test substance, and / or an optical signal representing the concentration of a soluble test substance. 11. The method according to any one of paragraphs. 1-4, in which the target introduced into the detection channel (12) is a combined target containing at least two components (41, 42), which are individually introduced into the detection channel (12). 12. The method according to any one of paragraphs. 1-4, in which either the soluble test substance forming the dissolved test substance (3), or the target (41; 41, 42), or both, contain a fluorescent marker capable of emitting an optically detectable fluorescent signal. 13. A system for determining the biological response of a target (41; 41, 42) to a soluble test substance, containing: a dispersion channel (11) having: the first entrance (111) into the dispersion channel for introducing the buffer liquid (2) into the dispersion channel (11), the second entrance (112) to the dispersion channel, located further than the first entrance (111) to the dispersion channel, for introducing the soluble test substance into the buffer liquid (2), flowing through the dispersion channel (11) to form the dissolved test substance (3) in the buffer liquid (2), and the exit (113) from the dispersion channel, located further than the first and second inputs (111, 112) into the dispersion channel, allowing the buffer liquid (2) containing the dissolved test substance (3) to exit the dispersion channel (11), - a pump (1) for creating a laminar flow of a buffer fluid through a dispersion channel (11), - an injector (7) of the test substance for introducing a soluble test substance into the laminar flow of the buffer fluid through the dispersion channel (11) to form a dissolved test substance (3) in the buffer fluid (2) having an initial concentration profile, which is then dispersed by the laminar flow of the buffer fluid (2) through the dispersion channel (11) so that a dispersed concentration profile is contained in the buffer liquid (2), - channel (12) detection, having: the first entrance (121) to the detection channel, which is arranged to communicate in fluid with the outlet (113) from the dispersion channel so that the laminar flow of the buffer liquid (2) exiting the dispersion channel (11) through the outlet (113) from the dispersion channel channel and containing the dissolved test substance (3) having a dispersed concentration profile, was directed through the first input (121) of the detection channel into the detection channel (12) so that in the detection channel (12) the dissolved test substance (3) forms the final a symmetric concentration profile of the dissolved analyte (3) in the buffer fluid, and at least one additional input (122, 123) into the detection channel for introducing the target (41; 41, 42) into the detection channel (12), at least one injector (8, 9) of the target for introducing the target into the detection channel (12) through at least one additional input (122, 123) into the detection channel for mixing with the dissolved analyte (3) having a finite symmetric concentration profile (33) in a buffer liquid (2) in such a way as to obtain a mixture having a combined concentration profile containing a target (41; 41, 42) having a constant concentration profile of a target mixed with a dissolved test substance (3) having a finite symmetrical end the centering profile of the dissolved test substance (3) in the buffer liquid (2), - means for holding in the detection channel (12) a mixture of the target (41; 41, 42) and the dissolved test substance (3) in the buffer liquid (2) so that at least one half of the combined concentration is retained in the detection channel (12) the profile contained in the buffer fluid (2), and - an optical detection unit (6), arranged and located for optical scanning in the channel (12) for detecting a mixture of the target (41; 41, 42) and the dissolved test substance (3) contained in the buffer liquid (2) held in the channel (12) detecting and containing at least one half of the combined concentration profile contained in the buffer liquid (2), for detecting, at various concentrations of the dissolved test substance (3) contained in the combined concentration profile, an optical a signal representing the biological response of the target to a soluble test substance, in which the output (113) from the dispersion channel and the first input (121) to the detection channel are connected to each other so that the laminar flow is maintained at the connection of these channels and in the detection channel. 14. The system of claim 13, wherein the inner wall (13) of the dispersion channel (11) at the exit (113) of the dispersion channel and the inner wall (14) of the detection channel (12) at the first entrance (121) to the detection channel have the same shape and size to ensure a continuous contour of the inner wall of the channel at the connection of the dispersion channel (11) and the detection channel (12). 15. The system according to claim 14, in which the inner wall (13) of the dispersion channel (11) at the exit (113) of the dispersion channel and the inner wall (14) of the detection channel (12) at the entrance (121) to the detection channel are made as a single the whole so as to form a common continuous inner wall (13, 14).

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